Efficient method of in-vitro micropropagation of piper longum

ABSTRACT

The present invention relates to an efficient method of in-vitro micropropagation of  Piper longum  plants. In particular, the present invention is directed towards the novel method for micropropagation of  Piper longum  from lateral bud (meristematic) explant (starting material), by culturing the explants on different medias. This method results in mass production of the plant  Piper longum  in a short span of time.

FIELD OF THE INVENTION

[0001] This invention relates to an efficient method of in-vitromicropropagation of Piper longum plants. In particular, the presentinvention is directed towards the novel method for micropropagation ofPiper longum from lateral bud explant (starting material), by culturingthe explants on different media.

BACKGROUND OF THE INFORMATION

[0002] Plants have been used for medicinal application ever since manbegan caring for his body and health. For centuries, the world hasdepended on the valuable properties of plant as a source of healing.Ayurveda, Siddha, Unani and Homeopathy continue to depend predominantlyon medicinal and aromatic plants as raw material for the formulation ofdrugs.

[0003] Medicinal Plants have attracted attention of not onlyprofessionals from various systems of Medicine, but also the scientificcommunity belonging to different disciplines. From time immemorial,medicinal plants are known and are being used since then. Based on theconsiderable knowledge about these plant species from the ancient books,the modern system of medicine have enabled these plants find a place inthe commercial market.

[0004] Medicinal plants are growing in importance day by day, because ofthe widespread interest in the adoption of multiple approaches to healthcare. Medicinal plants and products derived from them, have a uniqueplace in both preventive as well as curative medicine, as these plantsproduce an immense and diverse array of organic compounds, calledsecondary metabolites. Considerable efforts are being made all over theworld, to utilize more and more plant resources, as the medicine oftoday is found to shift from synthetic molecules to naturallysynthesized molecules. Also, these naturally synthesized molecules arebiologically more compatible and less toxic to human system as comparedto synthetics. The majority of natural products used medicinally inplants are secondary metabolites viz., terpenoids, steroids,cardenolides, quinine lignans, flavonoids or alkaloids. Medicinal plantshave gained pharmaceutical importance/therapeutic value due to itsspecific constituents/combination of secondary metabolites present inthem. These metabolites are often differentially distributed amonglimited taxonomic groups within the plant kingdom and participate ininteresting biological activities that can have high therapeutic value.

[0005] One such medicinal plant species that has gained pharmaceuticalimportance is Piper longum commonly known as long pepper. Piper longum,is a slender aromatic, perennial climber belonging to the familyPiperaceae, with thin and erect branches having sessile ovate leaves. Ithas minute dioecious flowers on spikes (inflorescence). Fruits, whichare used in medicine, are small red berries and often turns black onripening. It grows well in tropical and subtropical climate.

[0006] The compound of medicinal interest in Piper longum is present inthe female spike (inflorescence). Piper longum plant contains alkaloidpiperine as one of the active ingredient. Other active ingredientspresent in Piper longum plant are various amides viz. futoamide (Das &Kashinatham 1998), alkamide (Das & Kashinatham 1996), a new dimmer ofamide (Zang & Wang 1996), some minor amides (Kaul & Taneja 1998) and anamide alkaloid (piperlongumine).

[0007] The dry female spike of Piper longum is widely used in Ayurvedicand Unani system of medicine particularly for diseases of respiratorytract. It is a powerful stimulant of both digestive and respiratorysystem and has been shown to have rejuvenating effect on lungs. It isknown to play an important role in aiding the thermogenic response i.e.the result of increased thyroid hormone level in the body makes Piperlongum a typical Ayurvedic complementary component whose benefit is toincrease the bio-availability and to enhance the uptake of the otheractive ingredients. It is accepted as immuno-potentiating agent(Dahanukar 1991) and has shown promises as hepato-protective medicinalplant during clinical trials (Doshi et al 1994). The root of this plantis used for stomachache and is also prescribed in palsy, gout,rheumatism and lumbago.

[0008] Cultivation of Piper longum till recently was not very common.However, with increasing market demand of Piper longum, efforts arebeing diverted towards systematic cultivation of Piper longum. SincePiper longum is a shade loving creeper, majority of cultivators growthem as intercrop in coffee, arecanut & coconut plantation. Lateritesoil, soil rich in organic matter are suitable for cultivation of Piperlongum.

[0009] Conventionally, Piper longum is cultivated through plantingmatierals such as suckers, stem cuttings, seeds or rooted vine cuttingsin the month between March-April and later on transplanted in June atthe onset of monsoon.

[0010] The following are the drawbacks associated with conventionalmethod of cultivation of Piper longum using seed, suckers, rooted stemcuttings respectively.

[0011] Seeds as planting material: Very rarely seeds are used asplanting material, because number of viable seeds produced by one spikeis very limited. Since, female spikes are the medicinal part of theplant, available seeds material for plantation is very less or limited.Moreover, Piper longum seeds are highly heterozygeous in nature hence donot ensure quality homogenous raw material production. As seeds produceheterozygous plant (offspring), hence are not a good choice material.

[0012] Suckers as planting material: Suckers are produced only fromadult plants. They are produced after two years of plantation. Moreover,a plant produces very limited number of suckers, thus very small numberof planting material is available. This method is commercially not veryviable as, Piper longum has woody roots which develops in 2-3 years oldplant. From woody roots, few suckers develop which can be used forplantation. However, this method does not provide enough plantingmaterial.

[0013] Rooted stem cuttings as planting material: The most commoncultivation practice is from rooted stem cuttings. 8-10 cm long stemsegments are taken from the tip of the branches & artificially rootedusing rooting hormone. Only 70-80% cuttings develop rootings. These arethen transplanted in monsoon to the field with as much as 95% survival.The only snag with this method is that, when from a mature plant morethan 4-5 stem cuttings are taken, these plants show reduced yield andgrowth. It is noticed that if more than 10% plants are used from acommercial cultivation field for generating the cuttings, it severelyaffects the production of raw material.

[0014] The existing methods of cultivating Piper longum, is timeconsuming, as time is lost in preparing stem cuttings, rooted vines orsuckers for plantation of Piper longum. All this reduces the plantpopulation which plant otherwise have given female spike or roots havingmedicinal properties.

[0015] Contrary to this, the rate of multiplication by micropropagationis much higher and in a shorter time. Moreover, by micropropagation onecan develop clones as well as somaclonal variants for selection ofdesired traits.

[0016] Plant regeneration by tissue culture techniques is wellestablished. A wide variety of plant species has been successfullymicropropagated in vitro via organogenesis or somatic embryogenesis.Organogenesis leads to organ formation i.e. shoot or root, which can beisolated to induce development of roots or shoots, to produce fullplant. While somatic embryogenesis leads to the development of somaticembryos (embryos developed without fertilization) which have both shootand root initials and are capable of developing into whole plant.Although the ability of individual parts of plants and cells toregenerate into complete plants called totipotency, is a well knownphenomenon, each plant or plant part requires specialized studies toinvent the conditions that allow such regeneration. Some of the broadlyapplicable factors controlling growth and differentiation of suchcultures have been determined. The establishment of interactions amongdifferent groups of phytohormones and growth regulators alone or incombinations are responsible for certain interrelations existing amongcells, tissues and organs. There seems to be consensus that the successin inducing differentiation depends upon the type of explant,physiological condition of the explant and physical and chemical milieuof the explant during culture. Due to this, the science of tissueculture has been directed to optimize the physiological conditions ofsource plant, the type of explant, the culture conditions and thephytohormones used to initiate tissue culture. This substantiates thefact that development of a new process for proliferation of plants bytissue culture is not obvious.

[0017] There is no report on micropropagation of Piper longum fromlateral bud explant through direct rhizogenesis (organogenesis) followedby the plant regeneration. The scientist of the present invention havebeen successful in developing an efficient in vitro system ofmicropropagation of Piper longum from lateral bud explant through directorganogenesis.

[0018] There is an urgent need to develop a method which obviates thedrawbacks of the existing protocols. Looking at above mentioned problemsrelated to availability of planting material, it is imperative that thenovel method of producing elite, homogenous planting material isdeveloped. Keeping this in mind the present work has been undertaken.The present invention provides an efficient and cost effective method ofin-vitro micropropagation of Piper longum. By this method the rawmaterial of Piper longum produced is as high as 900 times theconventional methods, in less number of days.

OBJECTS OF THE INVENTION

[0019] It is an object of the present invention, to develop a highlyefficient, commercially viable micropropagation system of Piper longum,by in vitro culture or tissue culture.

[0020] It is an object of the present invention to select the explant,lateral bud (meristematic), and their isolation parameters from healthygrowing plants, under controlled condition.

[0021] It is yet another object of the present invention is to developsterilization system for surface sterilization of explants withoutdamaging the isolated tissues.

[0022] It is yet another object of the present invention is to developnutrient media requirement including growth regulators for (a) cultureinitiation from the explant (b) shoot bud proliferation on initiatedcultures and shoot elongation (c) rooting of regenerated shoots.

[0023] It is yet another object of the present invention is tostandardize the physical parameters viz. photoperiod, light intensity,relative humidity and temperature for all the stages of in vitroculture.

[0024] It is yet another object of the present invention is to find outthe time interval at which subculture of different culture stages couldbe performed.

[0025] It is yet another object of the present invention is to hardenthe in vitro cultured plants so that they can be acclimatized and safelytransferred from lab to land.

SUMMARY OF THE INVENTION

[0026] The present invention relates to an efficient method of in-vitromicropropagation of Piper longum plants. In particular, the presentinvention is directed towards the novel method for micropropagation ofPiper longum from lateral bud (meristematic) explant (startingmaterial), by culturing the explants on different medias. This methodresults in mass production of the plant Piper longum in a short span oftime.

[0027] The present invention involves in-vitro micropropagation of Piperlongum through direct rhizogenesis (organogenesis) by culturing lateralbud (meristematic) explant on various medias. Conventionally, in vitroinduction of rhizogenesis (organogenesis) is achieved from callusderived explants. Whereas, in the present invention, rhizogenesis isachieved directly from meristematic explant. The cells in lateral budsare meristematic i.e. these cells do not have alteration in their DNAsequencing hence a clonal offspring can be produced from them.

[0028] In accordance to the present invention, the meristematic cells ofthe lateral bud explant are transformed into root like appendages, byculturing in dark, on a culture initiation medium consisting of MS basalmedium, supplemented with a growth hormone, synthetic auxin NAA. Thegrowth hormone NAA is used for inducing rhizogenesis (root likestructure). These root like structures are subsequently grown in lightand once it reaches 1.5 cm in length, are cut into 0.5 cm long pieces.These long pieces are inoculated on growth hormone, cytokinin BAPcontaining nutrient medium. This growth hormone is used as shoot budinducing medium. The shoots regenerated from the shoot buds areelongated in shoot elongation medium supplemented with natural auxin,thereby promoting shoot elongation. The elongated shoots are thentransplanted to MS medium containing IBA for rooting of the shoots,thereby regenerating plantlets of Piper longum. These regeneratedplantlets are hardened and transferred to the field for cultivation in aconventional manner.

[0029] The plant hormone used are auxin and cytokinin of differentconcentrations.

[0030] The auxins are selected from 3-indole butyric acid (IBA),naphthalene acetic acid (NAA), 2,4-Dichlorobenzoic acid (2,4-D), IndoleAcetic Acid (IAA), Naphthoxy Acetid Acid (NOA) and the like. The mostpreferable auxin used in the present invention is NAA, IBA.

[0031] The cytokinins are selected from Kinetin, benzyl aminopurine(BAP), 2-iso-Pentenyl adenine (2-iP) and the like. The most preferablecytokinin used in the present invention is BAP.

[0032] The culture mediums for micropropagation of the plant Piperlongum are selected from MS basal medium (Murashige, T & Skoog. F(1962), a B5 medium (Gramborg, O. L. et. al (1968)) and the like. Themost preferable culture medium used in the present invention is MS basalmedium (Murashige, T. & Skoog (1962).

[0033] This invention has several advantages over the conventionalprotocols. The novel features of the invention are the following:

[0034] The method is simple and has a very high frequency of plantmultiplication in a short span of time and space.

[0035] The lateral bud explant selected from mature Piper longum plantsare found to respond efficiently, as high as 900 times the conventionalmethods.

[0036] The meristamatic cells by direct rhizogenesis produces clones.

[0037] The plants can be produced during all the seasons depending onthe requirement.

[0038] Nutrient media and growth regulator requirement for each stage ofmicropropagation (i.e. conversion of an explant into many plantlets) wasidentified and selected viz. culture initiation, rhizogenesis, inductionof adventitious shoot buds, shoot elongation and rooting.

[0039] Acclimatization and hardening of regenerated plants reduced themortality to 0% during transfer of plants to the field.

BRIEF DESCRIPTION OF DRAWINGS

[0040]FIG. 1 shows a flow diagram for the micropropagation of Piperlongum via direct rhizogenesis (organogenesis) derived from non sexualmeristematic explant tissues.

[0041]FIG. 2 shows a photograph of lateral buds inoculated on gelled MSbasal medium supplemented with auxin NAA, sucrose and myo-inositol.

[0042]FIG. 3 shows a photograph of induction of rhizogenesis fromlateral bud explants.

[0043]FIG. 4 shows a photograph of proliferation of shoot buds on MSbasal medium supplemented with cytokinin BAP and sucrose andmyo-inositol.

[0044]FIG. 5 shows a photograph of elongated shoots of Piper longum.

[0045]FIG. 6 shows a photograph of development of healthy roots fromregenerated shoots.

[0046]FIG. 7 shows a photograph of micropropagated plants which arebeing hardened in a portray in the green house having fan and pad systemfor cooling and foggers for maintaining relative humidity.

[0047] Specific description of applications of both somaticorganonogenesis procedure and the further development of mature plantsfrom somatic organonogenesis, are presented in the non limitingexamples.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Accordingly, the present invention provides an efficient methodfor micropropagation of Piper longum plant using lateral bud(meristematic) explant, said method comprising of the steps of:

[0049] A. Explant Collection and Surface Sterilization

[0050] Growing mother plant under controlled conditions in a green houseor shade house to ensure a healthy mother plant, a source of explants.

[0051] Plants were regularly irrigated and given monthly foliar spray ofa fungicide Bavistin (0.05-1%) and an insecticide Rogor (0.005-0.5%).

[0052] Giving an extra foliar spray of Bavistin (0.05-1%), two daysprior to collecting the explants.

[0053] Collecting the explants from a mother plant by cutting a twigwith a sharp scalpel, before 8 a.m. in the morning at a time when it hasthe highest turgor pressure, and immediately dipping the cut end intofungicide, Bavistin (0.05-1%) and bacteriacide, Ciprofloxacin solution(0.005-0.5%).

[0054] Washing the stem segment, after carefully removing the leaveswith a sharp scalpel, using a mild detergent and running tap water.

[0055] Finally, rinsing the explants with demineralized water anddipping them in autoclaved distilled water and transferring them underLaminar Flow benches (LF) for final surface sterilization.

[0056] Dissecting lateral buds from the stem segments under LF andtreating them with hydrogen chloride (HCl2) (0.002-0.2%) for 3 minutesand then giving three thorough rinses with autoclaved distilled water.

[0057] Further, surface sterilizing them by dipping in sodiumhypochloride (NaOCl) (0.5-3%) for 4 minutes and again giving threethorough rinses with autoclaved distilled water.

[0058] B. Inoculation on Culture Initiation Medium

[0059] Preparing culture initiation medium by using MS basal mediumdescribed by Murashige and Skoog in 1968. To the MS basal medium,sucrose (2-4%), agar powder (0.7-1.2%), 100 mg/L myo-inositol andNaphthalene Acetic Acid (NAA) (0.01-0.5 mg/L) was added. This medium wasautoclaved at 15 psi for 20 min and then cooled to solidify at roomtemperature.

[0060] Using sterilized forceps, each isolated and surface sterilizedlateral bud was inoculated in a test tube containing 15 ml ofabove-mentioned medium; by placing the cut end of the lateral budstouching the medium.

[0061] Forceps were flamed using absolute alcohol just before eachinoculation.

[0062] C. Incubation Incubating the inoculated explants in dark at 230C±10 C for 7-15 days. By this time, 6-7 root primordia appear from thebasal cut end of all the lateral buds.

[0063] D. Induction of Organogenesis (Rhizogenesis)

[0064] Transferring incubated cultures in light having 2000-4000 luxlight intensity.

[0065] A photoperiod of 16 hours was provided followed by a dark periodof 8 hours daily.

[0066] Allowing each root to grow to an average size of 3 cm in 45 days.

[0067] E. Shoot Bud Proliferation & Shoot Elongation

[0068] Cutting each root into 1 cm long segments aseptically under LF.

[0069] Sub-culturing the 18 root segments separately onto MS basalmedium supplemented with Benzyl Amino Purine (BAP) (0.5-3 mg/L), 2-4%sucrose, 100 mg/l myo-inositol and 0.7-1.2% agar for solidifying themedium.

[0070] Cultures were kept at 230 C±10 C under 2000-4000 Lux lightintensity for a photoperiod of 16 hours followed by a dark period of 8hours.

[0071] Each segment produced 50-55 adventitious shoot buds i.e.(18×50=900 shoot buds).

[0072] After 45 days each culture was cut into 2 pieces and each piecewas sub-cultured in a separate culture bottle containing 25 ml of thesame medium and growing them under similar physical conditions asmentioned above for shoot elongation for 45 days.

[0073] F. Rooting of Regenerated Shoots

[0074] 2.5-3 cm long shoots with 2-3 nodes were carefully separated fromthe culture clumps using a sterile scalpel under LF.

[0075] Sub-culturing 10 separated shoots in a wide mouthed bottlecontaining MS basal medium fortified with Indole Butyric Acid (IBA) (1-3mg/L), 2-4% sucrose, 100 mg/L myo-inositol and 0.7-1.2% agar.

[0076] Rooting of the regenerated shoots under reduced light 1000-3000Lux for 28 days. Photoperiod and temperatures remains the same asbefore.

[0077] G. Hardening of Regenerated Plantlets

[0078] Removing the micropropagated plants carefully from the agar.

[0079] Washing the roots under tap water to remove any trace of agaradhered to the roots.

[0080] Dipping roots in Bavistin (0.05-1%) for half an hour.

[0081] Planting the micropropagated plantlets in 1:1 ratio of soil andcocopeat.

[0082] Growing them in green house having 80% relative humidity (RH) andtemperature below 280 C for first week and then gradually increasing thetemperature to ambient temperature of 30-320 C and reducing the RH to40% over a period of 4 weeks.

[0083] Transferring hardened plants (for acclimatization to plantationsite conditions) to partial shade under shade-net for a week prior totransfer to the field.

EXAMPLE

[0084] Lateral bud (meristematic) explant from any position of young ormature plant of Piper longum were taken and cultured in MS basal mediumsupplemented with sucrose (2-4%), NAA (0.1-0.5 mg/l) and gelled withagar (0.7-1.2%). These were incubated in dark for 10 days at 23+1° C.and transferred to a light intensity of 1000-3000 lux for 16 Hrs.followed by 8 Hrs. dark period resulting into rhizogenesis, producing6-7 root like organs, by direct organogenosis from meristematic tissuesof lateral buds, which grew up to 1.5 cm in length by 45th day ofisolation.

[0085] The rhizogenic tissues were cut to 0.5 cm long segments withcytokinin BAP (0.5-3 mg/L) and solidified with agar (0.7-1.2%) resultingin adventitious shoot bud formation from all the root segments occurredon the same nutrient medium under similar physical conditions(2000-4000) lux light for 16 Hrs. followed by 8 Hrs. dark at 23+1° C.),the shoot buds elongated.

[0086] All separated shoots were responsive to IBA (0.1-3.1 mg/L) andproduced healthy root system from the basal cut ends of these shoot in 4weeks time.

[0087] Plantlets thus re-generated on transplant to in vivo on 1:1 soil& cocopeat in portrays having cavities showed good hardening. Hardenedplants were transferred to shad net for secondary hardening(acclimatization) to ambient field conditions for 1-2 weeks and theplantlets were transferred to field for cultivation in conventionalmanner.

[0088] The abbreviations used in the text have the following meaning: Asdefined herein NAA is Naphthalene Acetic Acid As defined herein IAA isIndole Actetic Acid As defined herein IBA is Indole Butyric Acid Asdefined herein NOA is Naphthoxy Acetic Acid As defined herein BAP isBenzylaminopurine As defined herein Kinetin is 6-furfuryl amino purineAs defined herein 2-iP is 2-iso-Pentenyl adenine As defined herein 2,4-Dis 2,4-Dichlorobenzoic acid As defined herein MS is Murashige & Skoog'smedium (1962) As defined herein B5 is Gramborg, O. L. et al's medium(1968) As defined herein RH is Relative Humidity

[0089] It is evident that these additional embodiments and variationswhich are not illustrated but are within the scope and spirit of thepresent invention. The above description and drawings are thereforeintended to be exemplary only and the scope of the invention is to belimited by the appended claims.

We claim:
 1. An efficient method of in-vitro micropropagation of Piperlongum from lateral bud explant, said method comprising the steps of:—collecting the lateral bud explant from the mother plant by cutting twigwith a sharp scalpel before 8.00 am and dipping the cut end into amedium comprising effective amounts of sterilants to keep the plantsfree from fungus and insect attack and transferring these plants underLaminar Flow benches (LF) for final surface sterilisation; cutting thelateral buds from the stem segments under LF and treating them withhydrogen chloride (HCl2) (0.002-0.2%) for 3 minutes and rinsing it withautoclaved distilled water; subjecting the lateral buds to surfacesterilisation by dipping in sodium hypochloride (NaOCl) (0.5-3%) for 4minutes and again giving three thorough rinses with autoclave distilledwater; inoculating the surface sterilised lateral buds in a cultureinitiation medium supplemented with growth hormones; autoclaving themedium of step iv, at 15 psi for 20 minutes and cooling it to solidifyat room temperature; incubating the inoculated explants in dark at 23°C.+1° C., for 7-15 days to produce 6-7 root primordia from the basal cutend of all the lateral buds; transferring the incubated cultures of stepvi, in light intensity of 2000-4000 lux and providing a photo period of16 hours followed by a dark period of 8 hours daily; allowing each rootto grow to an average size of 1.5 cm in 45 days; through directrhizogenesis (organogenesis); cutting each root of step viii, into 0.5cm long segments aseptically under LF; sub-culturing the 18 segmentsprocured from a single explant separately onto a shoot induction mediumsupplemented with the growth hormone; keeping the cultures of step x, at23° C. under light intensity of 2000-4000-lux for a photo period of 16hours followed by a dark period of 8 hours to produce 50-55 adventitiousshoot buds and 900 shoot buds (18×50); cutting each culture clump intotwo pieces after 45 days of interval and sub culturing each piece in aseparate culture bottle containing 25 ml of the same medium, and growingthem under similar physical conditions as mentioned step xi, for 45 daysfor shoot elongation; separating 2.5-3 cm long shoots with 2-3 nodesfrom the culture clumps using a sterile scalpel under LF; sub-culturing10 separated shoots of step xiii, in a wide mouth bottle containingrooting medium supplemented with growth hormone; rooting the regeneratedshoots of step xv, under reduced light intensity of 1000-3000 lux for 28days, with 16 hour photoperiod and temperature 23° C.+1° C.; removingthe micropropagated plants carefully from the agar contained in thesub-culturing medium; washing the roots of the micropropaged plants ofstep xvi, under tap water to remove any trace of agar adhered to theroots and dipping the roots in Bavistin (0.05-1%) for half an hour;planting the micropropagated plantlets in 1:1 ratio of soil andcocopeat; growing the above micropropagated plantlets in green househaving 80% relative humidity (RH) and temperature below 28° C. andgradually increasing the temperature to ambient temperature of 30-32° C.and reducing the RH to 40% over a period of 4 weeks for hardening theplants; transferring the hardened plants for acclimatization toplantation site conditions, to partial shade under shade-net for a weekprior to transferring the plants to the field; and transplanting themicropropagated hardened and acclimatized plants to the field forcultivation in a conventional manner.
 2. The method according to claim1, wherein the surface sterilants used are fungicide and bacteriacide.3. The method according to claim 2, wherein the fungicide used isBavistin (0.05-1%) and the bacteriacide used is ciprofloxocin(0.005-0.5%).
 4. The method according to claim 1, wherein the cultureinitiation medium used is MS basal medium (Murashige and Skoog in 1968),sucrose (1-5%), agar powder (0.2-1%), 100 mg/L myo-inositol supplementedwith growth hormone.
 5. The method according to claim 1, wherein thegrowth hormone used is auxin or cytokinin of various concentration. 6.The method according to claim 4, wherein the inoculating nutrient mediumis supplemented with growth hormone auxin.
 7. The method according toclaim 6, wherein auxin used is Naphthalene Acetic Acid (NAA) (0.01-0.5mg/L).
 8. The method according to claim 1, wherein the shoot inductionmedium comprises of MS basal medium, sucrose (1-5%), agar powder(0.2-1%), myo-inositol (100 mg/L) supplemented with the growth hormone.9. The method according to claim 8, wherein the shoot induction mediumis supplemented with growth hormone cytokinin.
 10. The method accordingto claim 9, wherein cytokinin used is Benzyl Amino Purine (BAP) (0.5-3mg/L).
 11. The method according to claim 1, wherein the rooting mediumcomprises of MS basal medium, sucrose (1-5%), agar powder (0.2-1%),myo-inositol (100 mg/L) supplemented with the growth hormone.
 12. Themethod according to claim 11, wherein the rooting medium is supplementedwith growth hormone auxin.
 13. The method according to claim 12, whereinauxin used is Indole Butyric Acid (IBA) (1-3 mg/L).